Leishmania RNA virus: when the host pays the toll

REVIEW ARTICLEpublished: 12 July 2012

doi: 10.3389/fcimb.2012.00099

Leishmania RNA virus: when the host pays the tollMary-Anne Hartley 1, Catherine Ronet1, Haroun Zangger1, Stephen M. Beverley2 and Nicolas Fasel1*

1 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland2 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, USA

Edited by:

Albert Descoteaux, INRS- InstitutArmand-Frappier, Canada

Reviewed by:

Dario S. Zamboni, Universidade deSão Paulo, BrazilMartin Olivier, McGill University,Canada

*Correspondence:

Nicolas Fasel, Department ofBiochemistry, University ofLausanne, Ch. des Boveresses 155,1066 Epalinges, Switzerland.e-mail: [email protected]

The presence of an RNA virus in a South American subgenus of the Leishmania parasite,L. (Viannia), was detected several decades ago but its role in leishmanial virulenceand metastasis was only recently described. In Leishmania guyanensis, the nucleicacid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting ahyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultantinflammatory cascade has been shown to increase disease severity, parasite persistence,and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were foundmostly in clinical isolates prone to infectious metastasis in both their human source andexperimental animal model, suggesting an association between the viral hyperpathogenand metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presentsas chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamedand notoriously refractory to treatment. Immunologically, this outcome has many of thesame hallmarks associated with the reaction to LRV: production of type 1 interferons, biastoward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminateparasites through oxidative stress. More intriguing, is that the risk of developing MCLis found almost exclusively in infections of the L. (Viannia) subtype, further indication thatleishmanial metastasis is caused, at least in part, by a parasitic component. LRV present inthis subgenus may contribute to the destructive inflammation of metastatic disease eitherby acting in concert with other intrinsic “metastatic factors” or by independently preyingon host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence mayprovide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis.Taking examples from other members of the Totiviridae virus family, this paper reviewsthe benefits and costs of endosymbiosis, specifically for the maintenance of LRV infectionin Leishmania parasites, which is often at the expense of its human host.

Keywords: Leishmania, Totiviridae, mucocutaneous leishmaniasis, dsRNA virus, toll-like receptor

INTRODUCTIONEndemic in 98 countries, leishmaniases are caused by vari-ous species of the Leishmania protozoan parasite and exhibit awide spectrum of clinical manifestations, ranging from a cuta-neous lesion (CL) to a fatal visceralization of disease (VL)(Kaye and Scott, 2011; Alvar et al., 2012). Parasites are trans-mitted through the bite of a sand fly vector, establishing infec-tion in a local CL, although asymptomatic infections are notuncommon. Some cases develop latently, reactivating later asa disseminated or metastatic infestation, complicating clinicaloutcome, and known to be refractory to standard therapeu-tic intervention. In South America, up to 10% of CL casesprogress to mucocutaneous disease (MCL) forming destruc-tive secondary lesions in the mucosa of the mouth and noseand even occurring in those with asymptomatic primary infec-tions. The risk of this clinical complication can be consideredas a distinguishing trait of the Leishmania (Viannia) subgenus,as it is mainly caused by species within the group (predomi-nantly L. braziliensis but also L. guyanensis and L. panamensis).Importantly, the clinical presentations of metastatic disease dif-fer between Leishmania species (Figure 1). For example, while

L. braziliensis and L. panamensis conform to the CL-to-MCL dis-semination pattern described above, L. guyanensis gives rise lessfrequently to mucosal lesions [although reported (Guerra et al.,2011)] and instead more often result in chronic disseminatedcutaneous leishmaniasis (DCL) with no reported anatomicalspecificity. Whatever the individual outcome, the general propen-sity toward infectious metastasis in South America seems to relyon an intrinsic parasite factor of the Viannia subgenus, wherespecies-specific features underlie tissue-specific divergences. Ashared feature among the metastatic Leishmania is their degreeof dormancy and chronicity, as reactivation and disseminationis often only developed months or even years after the ini-tial infection (Marsden, 1986; Ronet et al., 2010). This infectiveresurgence has been largely attributed to factors extrinsic to theparasite, such as the host environment and its genetic suscep-tibility, and it is proposed that virulent parasites are selected,kept dormant, and then later revived under immunosuppressedor stressed conditions. For example, antimony treatment dur-ing primary infection has been implicated in the developmentof MCL (Saravia et al., 1990; Arevalo et al., 2007; Souza et al.,2010). It is important to note, however, that dissemination is not

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Hartley et al. Leishmania RNA virus and virulence

FIGURE 1 | Leishmanial phylogeny and LRV presence. Intrinsic parasitefactors underlying clinical disparities in metastatic leishmaniasis. Specieslisted are those relevant to this text. The L. mexicana species complex (of theL. leishmania subgenus) was omitted for simplicity; thus far, no LRV has beenreported in their member species. CL, Cutaneous leishmaniasis; DCL,

Disseminated cutaneous leishmaniasis; VL, Visceral leishmaniasis; PKDL,Post Kala Azar dermal leishmaniasis; MCL, Mucocutaneous leishmaniasis;LRV, Leishmania RNA virus; (�), LRV found in numerous isolates withingroup; (��), LRV found only in a single, perhaps exceptional, isolate in thisgroup. Non-starred: groups in which LRV infection has not been reported.

completely unique to the Viannia subgenus. The donovani speciescomplex (of the Leishmania subgenus) shows a reversed symp-tomatic kinetic: visceralizing during a primary infection known asKala Azar with a risk of later reactivating as a disseminated cuta-neous infestation (Post Kala Azar Dermal Leishmaniasis, PKDL)(Figure 1). Still, the metastatic proclivity in these parasites isprobably quite different from that seen in MCL and the factorsunderlying visceralizing tropism in L. donovani are purportedlyencoded in species-specific genes (Zhang et al., 2008; Zhang andMatlashewski, 2010).

A common thread running through most cases of metastaticinfection is the onset of a destructive hyper-inflammatoryimmune response that is characterized by a deluge of activatedimmune cells, swelling, and destroying local tissue (Marsden,1986; Ronet et al., 2010). This overreaction is very likely insti-gated by a parasite factor. Indeed, the intrinsic Leishmania virusof L. guyanensis was recently shown to exacerbate inflamma-tion and may prove to be a major driver of metastatic poten-tial in L. (Viannia) parasites (Ives et al., 2011; Ronet et al.,2011). Although Leishmania viruses have been identified in majormetastatic strains of L. braziliensis and L. guyanensis, metastasiscan occur in absence of LRV, such as is the case for L. panamensis.Thus, LRV may have a variable contribution to this phenotype,acting alone or in concert with other factors, such as the hostgenetic background or species-specific parasite virulence factors.In this report, we review the current knowledge on Leishmaniavirus and its interaction with the host immune system in an effortto gauge its clinical impact and potential use in the diagnosis andtreatment of disseminated leishmaniases.

NEW WORLD LEISHMANIASIS AND Leishmania RNA VIRUSPARASITE FACTORS UNDERLYING DISEASE PHENOTYPEWhile the role of host factors cannot be overlooked, parasite pedi-gree is still the most reliable predictive tool of disease phenotype,

implying that heritable parasite factors are the major determi-nants of clinical variation. Yet, despite the considerable clinicaldifferences in leishmaniases across the Leishmania phylogenictree (Figure 1), unique genes between species are relatively scarce(Ivens et al., 2005; Peacock et al., 2007; Smith et al., 2007).Analysis of reference genomes for L. major, L. mexicana, L. infan-tum, and L. braziliensis have further confirmed the low number ofspecies-specific genes, albeit that variation amongst homologs isconsiderable (Rogers et al., 2011). Nevertheless, no obvious pat-terns emerge from this variation, which sufficiently explain thesymptomatic groupings of certain species. L. braziliensis standsout for having a high degree of single nucleotide polymorphismsand “lost” genes, which could, in conjunction with the presenceof its 67 unique genes, be the reason for its metastatic tropismand increased virulence (Rogers et al., 2011). Of particular inter-est are genetic differences involved in the control of oxidativestress. Being an intracellular infection, oxidative destruction inthe phagolysosome is a major mechanism of parasite elimination.Avoidance of this killing may be a way of developing latency andlater, metastasis. L. braziliensis is known to carry supplementarycopies of NADPH-dependent fumarate reductase and a homologof a glutathione peroxidase (as well as having lost a trypanoth-ione synthase-like protein) although it is not yet known whetherthese enzymes influence the sensitivity of L. braziliensis to oxida-tive stress. The sequencing of L. guyanensis and L. panamensisgenomes will add valuable information to the genetic determi-nants of oxidative resistance and their contribution to metastaticvirulence. Nevertheless, these genetic differences are still notsufficiently predictive or explanatory for the diversity of pathol-ogy. Instead, divergence in clinical outcome could be relatedto differential protein expression achieved through changes ingene regulation, copy number, or the presence of pseudogenes(Lynn and McMaster, 2008; Depledge et al., 2009; Rogers et al.,2011).






In light of LRV infection, parasitic genes controlling RNA-mediated interference (RNAi) are also of interest. The nucleic acidof LRV is potentially recognized by this parasite defense mech-anism targeting foreign RNA. While Leishmania are not knownto express RNA sensors such as those seen in mammals (PKR,RIG-I, MDA-5), some Leishmania species express a potent RNAiactivity (Lye et al., 2010). Functional RNAi machinery is mostlyabsent in the L. Leishmania subgenera (L. major, L. donovani,L. mexicana) but has been retained in the major metastatic para-sites of the L. (Viannia) subgroup (L. braziliensis, L. panamensis,and L. guyanensis) (Lye et al., 2010). Correspondingly, LRV1 hasbeen found in L. braziliensis and L. guyanensis, although thusfar not in L. panamensis (which has been less thoroughly exam-ined). The sole exception to this association is the presence ofLRV2 occurring in a single isolate of L. major (Scheffter et al.,1995), a Leishmania species a functional lacking RNAi (Lye et al.,2010). Variability in RNAi efficiency between evolutionary linesis also found in many other organisms (playing a strong rolein Drosophila and C. elegans, but minimally functional in mam-mals. Further studies are needed to define whether retention orlosses of RNAi are related to the evolution of viral interactionas is hypothetically exemplified in the co-maintenance of RNAiand LRVs.

LRV: A MEMBER OF THE Totiviridae FAMILYLeishmania viruses are classified in the Totiviridae family(Patterson, 1990; Weeks et al., 1992) encompassing non-enveloped, icosahedral particles present in protozoa [T. vaginialisand G. lamblia (Wang and Wang, 1991)], yeast (Wickner, 1996),fungi, plants, arthropods (Wu et al., 2010; Zhai et al., 2010;Isawa et al., 2011), penaeid shrimp (Poulos et al., 2006) and evenvertebrates [salmon (Lovoll et al., 2010)]. The 40 nm viral par-ticle is composed of a non-segmented dsRNA genome between4 and 8 kb in length encoding a major capsid protein and acapsid-RNA-dependent RNA polymerase (RDRP) fusion pro-tein, essential for the replication of the dsRNA virus (Figure 2A).

This RDRP has, however, been observed as independent fromthe capsid protein (Figure 2B), for example in the myonecrosisvirus infecting penaeid shrimp (Poulos et al., 2006) and the fun-gal virus, Helminthosporium victorivirus (Huang and Ghabrial,1996). Some totiviruses have additional proteins encoded in theirRNA genome, such as the antifungal killer toxin that was used toprotect maize against corn smut (Allen et al., 2011). LRV seemsto follow the generic totiviridae conformation described above,albeit for gene arrangement and sequence variation betweenLRV1 and LRV2, described below.

While virus-like particles were described in Leishmania her-tigi in 1974 (Molyneux, 1974), the first molecular description ofLeishmania RNA virus came only in the subsequent decade for thetwo L. guyanensis strains: MHOM/SR/81/CUMC1A (Tarr et al.,1988; Stuart et al., 1992) and MHOM/BR/75/M4147 (Widmeret al., 1989) then later in L. braziliensis (Salinas et al., 1996).The sole reported LRV found outside Viannia was identified inthe L. major strain (MHOM/SU/73/5-ASKH) (Scheffter et al.,1995). These have been separately categorized as LRV1 and LRV2in L. (Viannia) and L. major, respectively, due to their signifi-cant sequence differences. Phylogenetic studies on LRVs showedthat the genetic distances between LRV1 and LRV2 are similar tothose between each parasite strain and that this similarity wasfurther clustered according to geographical origin of the para-site (Scheffter et al., 1995; Widmer and Dooley, 1995). Thus,the viruses were present in Leishmania parasites prior to theNew/Old World divergence and seem to have co-evolved withtheir Leishmania host (Scheffter et al., 1995; Widmer and Dooley,1995). Unlike LRV1 of L. (Viannia), the relationship of LRV2 inL. major to disease severity or alteration in clinical phenotype hasnot yet been explored.

Thus far, no dsRNA viruses have been identified inLeishmania’s protozoan contemporaries such as Trypanosomabrucei, T. cruzi or Plasmodium. The most studied members ofthe Totiviridae family are the two dsRNA L-A and L-BC virusesinfecting S. cerevisae (Wickner, 1996), where studies focus on

FIGURE 2 | Organization of the Totiviridae genome. (A) Type I representsthe classic capsid-dependent organization with overlapping ORFs, wherea −1 or +1 frameshift (under the control of a pseudoknot structure) allows for

translation of the RDRP fused to the capsid. (B) Type II hosts acapsid-independent RDRP ORF. ORF, open reading frame; RDRP,RNA-dependent-RNA-polymerase; IRE, Internal ribosomal entry site.






their relation to the host and modulation of gene expression. Asdetermined in S. cerevisae, the plus-ssRNA is synthesized on adsRNA template by RDRP. Interestingly, these viral ssRNAs lackthe 5′ cap structure and are not polyadenylated, features essentialfor mRNA stability and efficient translation. It has been sug-gested that this vital 5′ cap could be pirated from host RNA bya unique mechanism dubbed “cap-snatching”. Here, the 5′ m7Gpof host mRNA is transferred onto the diphosphorylated 5′ endof the viral transcripts (Fujimura and Esteban, 2011). The viralcapsid plays a central role in this theft, where a histidine at posi-tion 154 in the protein has been deemed essential. Further, thepresence of a trench on the capsid’s outer surface [identifiedby crystallography (Naitow et al., 2002)] is reminiscent of thosedescribed for yeast guanyltransferases suggesting convergent evo-lution (Fujimura and Esteban, 2011). Cap-snatching could also becarried through to fungal totiviruses, as evidenced by some con-served amino acids in the proposed catalytic cleft (Fujimura andEsteban, 2011). It is not known whether this type of capping isalso relevant in other totiviruses or if they rather use the “decap-ping” mechanism previously proposed for yeast L-A viruses (Tanget al., 2005) where unprotected viral mRNAs are shielded fromhost cell exonucleases by allowing translation of the viral ssRNAthrough an internal ribosomal entry site (Masison et al., 1995).Besides mRNA cap-theft, the translational toll of the virus on thehost cell can be extraordinarily taxing. In yeast, viral capsid hasbeen shown to comprise several percent of total host protein, pro-ducing at least 1000 particles per cell, each particle consisting ofabout 120 capsid proteins (but only 1 or 2 capsid-RDRP fusionpolypeptides).

Translation of viral ssRNA has been best described in yeast.Here, Totiviridae translation takes place in the host cytoplasmproducing the capsid and in most instances, a capsid-RDRPfusion polypeptide. This latter protein is obtained through aninefficient −1 or +1 frameshift, which is under the control of anRNA pseudoknot structure placed upstream of the capsid genestop codon (Figure 2). N-terminal acetylation of the capsid pro-tein is essential for viral assembly with the capsid being, in turn,important for the packaging of the RDRP and viral genome intothe particle, whereas the polymerase domain of RDRP is requiredfor capturing the ssRNA viral molecule (Ribas and Wickner, 1998;Fujimura and Esteban, 2011). Motifs important for polymerasefunction are conserved among the RDRP sequences of differ-ent members of the Totiviridae (Maga et al., 1995b; Routhierand Bruenn, 1998), suggesting similar mechanisms for theirtranscription.

Similarly to other totiviruses, LRV exists predominantly as a5.3 kb double-stranded RNA within its capsid, having a plus-strand mRNA for viral polypeptide synthesis (Weeks et al., 1992).The L. guyanensis viral particles were demonstrated to have RNApolymerase activity, essential for the replication of RNA viruses(Widmer et al., 1990). Comparison of the two genomes revealeda consensus nucleotide sequence of 5283 base pairs with an over-all 76% sequence identity (Scheffter et al., 1994). Each guyanensisvirus was given a different designation, namely LRV1-1 (for thevirus of the L. guyanensis CUMC-1) and LRV1-4 (L. guyanensisM4147) although a revision of this nomenclature is currently inpreparation. Further analysis of LRV1 sequences identified 3 open

reading frames (ORFs) on the plus-strand of LRV1-1 while 4ORFs were identified in LRV1-4. In both cases, ORF2 and ORF3are known to encode the major viral proteins of the capsid andcapsid-RDRP. Similar to other totiviruses, this RDRP is formedas a fusion protein by a +1 ribosomal frameshift (Maga et al.,1995a; Lee et al., 1996; Ro et al., 1997b; Kim et al., 2005). The pre-dicted protein sequences of the other ORFs have, so far, shownno significant homology with known proteins or any evidenceof encoding polypeptides (Stuart et al., 1992; Scheffter et al.,1994). In the case of LRV-2 (the exceptional LRV occurrencein “Old world” leishmanial parasites), the site encoding RDRP(ORF3) is predicted not to be overlapping that of the capsid(ORF2) but is rather separated by a single codon and thereforecould be encoded in an independent ORF. Here, translation ofthe RDRP gene in LRV-2 could be driven by the presence of anadditional internal ribosomal entry site upstream of ORF3 or viathe action of a pseudoknot structure participating in ribosomal“hopping,” as proposed by Scheffter (Scheffter et al., 1995). Theselatter mechanisms could be relevant even for those LRV genomes,which do have overlapping ORFs, but the extent to which theseare functioning have not yet been resolved. Alternatively, wecould postulate that RDRP is synthesized from a trans-splicedmRNA, as done for mRNAs in trypanosomatids. However, sucha trans-splicing mechanism would generate mRNAs of smallersizes (not observed thus far) and would hinder the productionof capsid-RDRP fusion proteins.

The fact that, in Leishmania, mRNAs are matured by theaddition of m7GpppX through trans-splicing of a capped39-nucleotide mini-exon sequence, suggests that translation ofparasitic (non-viral) ssRNA could rely on internal ribosomalentry (Zamora et al., 2000). Interestingly, an internal ribosomalentry site has been mapped in the LRV-1 genome and wouldpotentially allow for the translation of uncapped viral ssRNA(Maga et al., 1995b). The occurrence of cap-snatching, its con-tribution to viral plus-strand stability and quantitative effects ontranslation remain to be determined.

THE TOLL OF LRV INFECTION: FROM PARASITE FITNESS TO HUMANDISEASEIn pathogenic microbes, signs of totivirus infection could be dis-played as alterations to fitness and virulence. For example, viralpresence in yeast and fungal species (Wickner, 1996; Schmitt andBreinig, 2006) offers a survival advantage through the expres-sion of a toxin that kills their uninfected peers. Contrarily, viralinfection of certain fungi (Beauveria bassiana and Bostrytis cinera)seems to reduce their virulence and thus their efficacy in the bio-control of agricultural pests (Castro et al., 2003; Dalzoto et al.,2006). More commonly, however, totiviral infections do not showsignificant phenotypic alterations or pathology. This, along withtheir widespread distribution in protists, plants, arthropods, andpossibly fish suggests that Totiviruses could be more abundantthan estimated. Given the increasing number of metagenomicand “pathogen discovery” projects, we may soon be able to bet-ter estimate the prevalence of Totiviridae and thus determine itstoll on host evolution.

Thus far, only few studies have reported on the impact ofTotiviridae on overall mRNA turnover and protein expression in






the host cell. In T. vaginalis, the presence of a dsRNA virus causessignificant differences at the protein level between infected andnon-infected varieties, including quantitative and qualitative dif-ferences in cysteine proteinases known to modulate T. vaginalispathogenesis (Provenzano et al., 1997). Most provocative andstill to be confirmed is the up-regulation in synthesis and sur-face expression of the immunogen P270 (Khoshnan et al., 1994),which may impact pathogenesis in the human. Other members ofthe Totiviridae family have been described as directly pathogenic.The myonecrosis virus, for example, was shown to cause cell deathin the skeletal muscle of the Pacific white shrimp (Lightner et al.,2006; Poulos et al., 2006) while a new member of the Totiviridaefamily has been implicated in Atlantic salmon cardiomyopathysyndrome (Lovoll et al., 2010). This latter virus was classifiedthrough amino acid sequence alignment of capsid-RDRP pro-teins, showing the closest match with the Giardia lamblia virusand stands as the first example of a totivirus directly infecting amember of the metazoa (Lovoll et al., 2010).

As already introduced in this review, a particularly interestingrelationship exists between the dsRNA virus of Leishmania par-asites and the human host, having important consequences onthe clinical outcome of leishmaniasis. LRVs have been detectedin both active and healing lesions or scars, confirming LRV pres-ence in field isolates (Cadd et al., 1993; Salinas et al., 1996; Saizet al., 1998; Ogg et al., 2003) although the their prevalence andclinical significance is not yet known. In Leishmania, the role ofLRV on parasite metabolism and gene expression has not beenstudied in detail and there is no information on why some para-site strains are able to maintain LRV and others not. While RNAimay serve to cull the viral herd in Leishmania (thereby allowingtheir persistence through Malthusian fitness), a range of enzymesand proteases potentially able to aid viral replication can also befound in Leishmania parasites. A specific cysteine protease (possi-bly a homolog of LmjF08.1040 according to our database search)has been implicated in the processing of the viral capsid-RDRPprecursor (Carrion et al., 2003). On the other hand, the LRV cap-sid protein possesses an endoribonuclease that cleaves uncapped,non-polyadenylated, plus-strand, viral mRNA at a single site inthe 5′-untranslated region, which was proposed to also cleavedecapped, non-polyadenylated mRNAs belonging to the parasite(MacBeth and Patterson, 1995, 1998; Ro et al., 2004).

While molecular studies on LRV continued more or less con-stantly since its description, the interest in its functional andclinical role remained low. Several groups speculated an influ-ence of LRV presence on parasite virulence, but over 10 yearspassed with no major studies on the biological impact of LRV1 onLeishmania parasites and no efforts to analyze isogenic or clonallines at a protein level. An interest in LRV as a determinant of vir-ulence resurfaced only in 2011 through the use of L. guyanensisclones isolated from human patients (Ives et al., 2011). The iso-lates were previously classified by differing metastatic proclivities,ranging from highly metastatic (M+) to non-metastatic (M−)as seen in the golden hamster model (Martinez et al., 1991). Inthis study, they compared soluble proteomes from promastigotesand revealed that M+ and M− clones express distinct acidic andneutral isoforms of cytosolic tryparedoxin peroxidase (cTXNPx).This differential expression was conserved in L. (Viannia) isolates

from cutaneous (M−) vs. mucosal (V+) lesions and may relateto the mechanisms by which the activity of cTXNPx is modulatedand/or the gene product(s) are post-translationally modified. Themetabolic role of cTXNPx in oxidative stress reiterates the impor-tance of antioxidant defense in the development of MCL andfurther endorses the cTXNPx gene as a unique factor underly-ing the development of metastatic infection. Further differencesin cTXNPx activity were viewed under oxidative stress and dur-ing infection. Upon H2O2 treatment or heat shock, cTXPNx ismostly detected in a dimerised form in M+ L. guyanensis andL. panamensis strains, while it is mostly undimerized in M− par-asites. These data provide evidence that protection to the hostile,oxidative environment encountered in the host cell by Leishmaniapromastigotes could be linked to cTXPNx conformation andmay be relevant to intracellular parasite survival and persistence,which are prerequisites for the development of metastatic disease(Acestor et al., 2006; Walker et al., 2006). Whether survival advan-tages of metastatic Leishmania in stressful situations (Figure 3)are due to a direct action of LRV on parasite metabolism or indi-rectly by an action via the host innate immune response meritsfurther investigation.

MAINTENANCE OF LRV INFECTIONIt is unknown how LRV1 is maintained and transmitted in theL. (Viannia) subgenus. Extracellular transmission of Totiviridaeis rare, albeit documented for the G. lamblia virus, which caninfect virus-free hosts (Ro et al., 1997a). Within the Totiviridaefamily, viral spread is either vertical (from mother to daughter)or horizontal [by cell fusion during mating and hyphal anasto-mosis (Dalzoto et al., 2006)]. Infection of virus-free Leishmaniaparasites has failed or lasted only transiently (Armstrong et al.,1993), a fate similarly encountered by workers studying otherTotiviruses. Thus, extracellular transfer of the dsRNA virus isnot likely to occur in Leishmania. In fungi, transmission takeplaces during genetic exchange and may apply to leishmania(Ravel et al., 1998; Nolder et al., 2007; Odiwuor et al., 2011). It isonly recently that exchange was experimentally demonstrated inL. major via a sexual cycle in the sand fly stage (Akopyants et al.,2009). However, unlike yeast and fungi, this form of mating (andthus viral exchange) is highly infrequent in Leishmania parasites,perhaps explaining the low prevalence of LRV across the genus.

Besides a single report on the generation of LRV- parasitesfrom an LRV+ parent (Ro et al., 1997a), a stable LRV infec-tion into naïve parasites has not yet been demonstrated. Thislone observation has proven difficult to reproduce in our labo-ratories, and may be the result of a fortuitous event reflectingnatural variation of LRV virus levels as well as its loss (Iveset al., 2011). Nonetheless, these isogenic LRV+ and LRV− strainshave since been used to study the role of RNAi machinery (Lyeet al., 2010) as well as that of LRV in disease outcome (Iveset al., 2011; Ronet et al., 2011). Likewise, there has been noreported success in the development an infectious system withwhich to stably reintroduce LRV into LRV-deficient lines. It ispossible that the RNAi machinery itself is the obstacle imped-ing viral introduction, as subgenus Viannia parasites (but not“higher” Leishmania) possess a potent RNAi pathway (Lye et al.,2010). A recent study with the yeast L-A virus suggested that






FIGURE 3 | Summary of possible survival advantages in Leishmania (Viannia) parasites associated with LRV infection. Despite the metabolic andtranslational toll on the host, LRV infection is associated with some benefits in survival advantages for its host parasite.

reintroduction of an active RNAi pathway in this species wasincompatible with maintenance of the dsRNA virus (Drinnenberget al., 2011) and correspondingly, the absence of this pathwayin S. cerevisae could explain why they carry such taxing quan-tities of virus. The interplay between dsRNA viruses and RNAihas been suggested as a force contributing to the loss of RNAiin both Leishmania and S. cerevisae (Beverley, 2003; Robinsonand Beverley, 2003; Drinnenberg et al., 2011). More research isneeded to assess the toll of LRV presence on parasite pathogenic-ity and what benefits may be gained by the maintenance of suchan infection.

IMMUNE RESPONSE IN MCL PATIENTSTHE Th1/Th2 DOGMAIn animal models of cutaneous leishmaniasis caused by L. major,the immunological dogma correlates resistance to disease withthe development of a CD4+ Th1 response, and susceptibilitywith a CD4+ Th2 response (Figure 4A). Th1 cells are character-ized by the production of IFN-γ and lymphotoxin whereas Th2cells classically produce IL-4, IL-5, and IL-13. In humans and incutaneous leishmaniasis caused by species other than L. major,although the protective parameters are similar, the response isnot as polarized as reported in mouse models. Here, the responsemore graded and heterogeneous, albeit still harboring a pre-dominant CD4+ Th1 type response upon healing (either aftertreatment or spontaneously) and having a Th2 cytokine pro-file of IL-4 and IL-13 in non-healing phenotypes. Protectionagainst L. major has been correlated to early IL-12 productionby dendritic cells and macrophages, which in turn induces IFN-γproduction by Natural Killer (NK) cells and at a later point, by

Th1 cells. However, the levels of IFN-γ do not always correlatewith resistance, as similar levels of this cytokine were observedfollowing leishmanization, independently of lesion development.Tumor necrosis factor alpha (TNF-α) and IFN-γ act synergis-tically to induce nitric oxide synthase (iNOS) in macrophages.This enzyme catalyzes the synthesis of citrulline and nitric oxide(NO) from arginine leading to the killing of intracellular amastig-ote parasites. Arginine, however, is also the substrate of arginase,producing the polyamines necessary for parasite growth andmay even be used by the parasite in immune evasion [fora review on L-arginine metabolism and Leishmania infectionsee Wanasen and Soong (2008)]. For example, it was shownthat Leishmania arginase could subvert iNOS-dependent killingby reducing macrophage L-arginine (Gaur et al., 2007). TheTh1/Th2 paradigm further disregards the influence and inter-action of other, more recently described, T-cell subsets, such asTh17, vilified as the architect of chronic destructive inflammationsuch as that seen in metastatic leishmaniasis, but nonetheless, hasstood as the most reliable guide in the prediction of parasitotoxicimmune responses.

Challenging the dogma is the response seen in MCL, wherehigh levels of Th1 pro-inflammatory cytokines (TNF-α andIFN-γ) are associated with T cell hyperactivity and a worsen-ing of the disease (Carvalho et al., 1985; Silveira et al., 2009).Furthermore, patients who are refractory to treatment anddemonstrate an inability for their cytokine profile to switch froma mixed Th1/Th2 to a predominantly Th1 type (Pirmez et al.,1993; Diaz et al., 2002), still have elevated levels of TNF-α in theirlesions. A common immunological trait in MCL is a decreasedresponse to Th1-suppressing cytokines (IL-10 and TGF-β)






FIGURE 4 | Immunological Th1/Th2 dogma in leishmaniasis.

(A) Cutaneous leishmaniasis (CL) shows a simple, graded bipolarphenotypic range while (B) metastatic/mucocutaneous leishmaniasis(MCL) shows its third phenotype in a Th1 extreme. NO, nitric oxide.

(Bacellar et al., 2002). Thus, while in CL, tipping the Th1/Th2balance in either direction results in a simple, graded, bipolarhealing vs. non-healing phenotype (Figure 4A), MCL displays itsthird phenotype as a deleterious pro-inflammatory Th1 extreme(Figure 4B). This response is often not only “extreme” in its quan-tity of inflammatory mediators, but also in its persistence overtime. In this archetypal MCL, increased levels of TNF-α, CXCL10,and CCL4 within a mixed intra-lesional Th1/Th2 response,results in an emphasized cytotoxic T cell activity, which mayunderlie the localized tissue damage and development secondarylesions that characterize the pathology of MCL (Pirmez et al.,1993; Faria et al., 2005; Gaze et al., 2006; Vargas-Inchausteguiet al., 2010). What pushes the host immune system into thisdeleterious Th1 extreme may be related to either a genetic hyper-activity within the host or, indeed, the presence of a potent innateimmunogen in the parasite, such as LRV1 dsRNA.

The cytokine triggers detonating infectious metastasis mightalso be related to a loss of control. Indeed, as mentioned above,MCL lesions often have diminished expression of IL-10R, ren-dering them insensitive to its anti-inflammatory role that usuallyworks to maintain an appropriate response by dampening thetorrent of stimulatory signals in the lesion environment. Type IIFNs work in a similar manner, down-regulating IFNγ-R on thesurface of macrophages and thus rendering these cells insensi-tive to intracellular pathogen killing via reactive nitrogen species(Rayamajhi et al., 2010). Another possible trigger may be an earlypeak of IFN- β. Early IFN-β in models of metastatic leishmaniasishas been reported to chronically modulate the immune responseand parasite killing, favoring parasite survival and infectious

dissemination to different organs [as was shown for the dissemi-nation of L. braziliensis in TNF-α−/− mice (Rocha et al., 2007)].Indeed, mice deficient for the IFN-β receptor (IFNAR) were pro-tected from leishmaniasis, presenting with smaller lesions andreduced antigen-specific and Th1 immune responses after infec-tion with L. amazonensis (Xin et al., 2010). In addition, increasedand sustained neutrophil recruitment in IFNAR−/− mice partic-ipate to boost parasite killing. Consequently, type I interferonsand molecules of its signaling pathway may emerge as therapeutictargets in infections with species of New World Leishmania.

INNATE IMMUNITY TO Totiviridae: TLRs, RLRs AND NLRsThe innate immune response to Leishmania parasites has been thetopic of recent reviews, which summarize TLRs (Liese et al., 2008;Faria et al., 2012; Singh et al., 2012). In the case of the L. (Viannia)subgenus, this has been exemplified for TLR2 in L. panamensisand L. amazonensis (Gallego et al., 2011; Vivarini Ade et al.,2011). Outside our recent description in Leishmania guyanensis,viral endosymbionts as innate immunogens have not yet beendescribed. The immunogenicity of their molecular componentscould reveal many potential pathways to influencing the humanimmune response. The nucleic make-up of Totiviridae (dsRNA)is sensitively recognized by a variety of pattern-recognition recep-tors in immune cells. In our report, LRV recognition occurredthrough the TLR3 pathway. TLR3, is well-known to recognizedsRNA, producing an IFN-β mediated anti-viral response, whichwe proposed as underlying the exacerbated disease phenotype.Indeed, TLR3−/− mice showed no LRV-mediated increase indestructive inflammation. Interestingly, its ssRNA-recognizingsister receptor, TLR7, is also stimulated by LRV components(as seen by a slight up-regulation of pro-inflammatory cytokinesin in vitro macrophages) but did not extend to a clinical role in thein vivo mouse model (Ives et al., 2011; Ronet et al., 2011). The roleof the last endosomal TLR recognizing DNA (TLR9) has not yetbeen reported on. It should be pointed out that dsRNA can alsobe detected by a variety of non-TLR pathways. For example, Rig-Like-Receptors (RLRs) are stimulated through binding either the5′ RNA triphosphate (RIG-1) or whole dsRNA such as poly I:C(MDA-5) and both converge to mediate IFN-β dependent inflam-mation. Further, this nucleic acid is also known to stimulatethe Nod-Like-Receptors (NLRs) through an unknown mecha-nism, sparking inflammasome activity and thus may be anothermechanism of immune disruption and perhaps even local tissuedamage. Whether or to what extent these RLR and NLR pathwaysare involved in metastatic leishmaniasis is still unknown. Theseinnate pathways peak our interest in the primary host-parasiteencounters and we predict that pattern recognition in epithelialand neutrophil cells will attract particular attention in the comingyears. This potential for immunogenicity amongst totiviruses, isnot only important for the disease pathogenesis of their pathogenhosts, but also serves to single-out these pathogen symbionts asunique molecular targets for use in diagnostic and therapeuticstrategies.

THE OXIDATIVE RESPONSE IN MCLDespite the similarity between LRVs in the L. (Viannia) subgenus,there are still major variations in metastatic phenotype amongst






infections from different LRV+ parasite isolates. It is possiblethat differences in sensitivity to oxidative stress could moti-vate these deviations, prolonging parasite survival or triggeringthe latency, which underlies many recurrent infections. Severaloxidative mediators could participate in Leishmania killing suchas NO (produced by iNOS from L-arginine), H2O2 (detoxifiedin mammalian cells by catalase or glutathione peroxidase) andcytoplasmic superoxide as well as its byproduct, peroxinitrite,[generated mainly by NADPH oxidase at levels controlled bycytoplasmic superoxide dismutase (SOD1)]. Even though contra-dictory results have been reported on the roles of NO, ROI (Rochaet al., 2007; Khouri et al., 2009), and the regulation of arginase(Muleme et al., 2009), it is generally accepted that they are keymechanisms in the elimination of leishmanial infection. The sameview is held for LRV-infected parasite species such as L. guyanen-sis, albeit that the role of NADPH oxidase is still matter of debate(Rocha et al., 2007). Oxidative evasion could certainly be achievedthrough parasitic manipulation of arginase I, a key enzyme in theproduction of urea (instead of parasitotoxic NO) from arginine.This enzyme is thought to be antagonized by IFN-γ (Menzieset al., 2010) and under control of Th2 cytokines (IL-4, IL-10,and IL-13) (Modolell et al., 1995). This latter regulatory mech-anism has since been challenged (Muleme et al., 2009). Indeed,metastatic L. braziliensis species were reported to induce higherlevels of insulin-like growth factor (IGF), a substance known toup-regulate arginase activity (Vendrame et al., 2010). None ofthese reports, however, have addressed the possible influence ofLRV infection on oxidative resistance.

As previously mentioned, the recurring concurrence of resis-tance to oxidative stress in metastatic parasites seems more thanfortuitous. Interestingly, however, these parasites also seem toinduce a higher level of the oxidative stress to which they areresistant, begging the question of: which causes which? For exam-ple, the metastatic L.g. M5313 clones persist in activated bonemarrow derived macrophages (BMMφ) despite an elevated NOlevel (Acestor et al., 2006), shown to be the result of an up-regulation in the iNOS gene (Ives et al., 2011; Ronet et al., 2011).We reported that L. guyanensis cytoplasmic tryparedoxin peroxi-dase (cTXPNx) could be involved in this protection, as it seems toincrease resistance to H2O2 in metastatic parasites (Acestor et al.,2006). Conversely, rapidly healing skin lesions are also associatedwith increased iNOS expression as well as IFN-γ, IL-12 and theirsignal transducer, STAT4 (Rocha et al., 2007). This latter groupof molecules was described as essential in the healing of L. majorinfection, whereas only IFN-γ and STAT4 (but not IL-12) wereessential in the healing of L. mexicana infection (Buxbaum et al.,2002).

Khouri et al. showed that the elimination of L. braziliensiscould depend on superoxide (and not NO) (Khouri et al., 2009)and further that exogenously added IFN-β impairs superoxidekilling, favoring parasite survival by up-regulating cytoplasmicSOD1 (Khouri et al., 2009). These data suggest that productionof IFN-β could increase susceptibility of the host to infection [fora review see (Trinchieri, 2010)]. It is here where the presenceof an intra-parasitic dsRNA virus becomes especially importantin the disease process. Viral dsRNA is innately recognized byTLR3, and known to induce the secretion of IFN-β and other

pro-inflammatory mediators. Already, several murine models ofinfection have shown that early TLR3-induced IFN-β secretiondoes not play its expected anti-viral role but is rather involvedin the eruption of a pathological immune response. Indeed,TLR3−/− mice, in these infections, have an increased survivalrate when compared to their wild type (Lang et al., 2006; LeGoffic et al., 2006; Cavassani et al., 2008). Further, TLR3 ligationup-regulated pro-inflammatory mediators (such as IFN-β, TNF-α, IL-6, and various chemokines) that promoted organ damagewith a high dependency on type I IFNs (specifically IFN-β),CD8+ cytotoxic T cell and NK cell activity (Lang et al., 2006;Le Goffic et al., 2006; Cavassani et al., 2008). Further evidence ofthe destructive influence of type I IFNs has been observed whenblocking these cytokines in vivo (Sakaguchi et al., 2003; O’Connellet al., 2004; Lang et al., 2006; Baccala et al., 2007; Rayamajhi et al.,2010). These studies also demonstrate that type I IFNs modulatethe Th1/Th2 polarization of the immune response via the inhi-bition IL-12 expression, impairing DC maturation, decreasing Tcell IFN-γ production and down-regulating IFNγ-R on myeloidcells (O’Connell et al., 2004; Lang et al., 2006; Baccala et al., 2007;Cavassani et al., 2008). The importance of oxidative stress in con-trolling L. (Viannia) parasite load and its immune response isevidently not understood and studies have yet to account for theimpact of Leishmania dsRNA virus on NO and superoxide level.

EXPERIMENTAL ANIMAL MODELS OF MCLThe golden hamster is possibly the best animal model in whichto study metastatic leishmaniasis caused by the L. (Viannia)guyanensis, panamensis, and braziliensis (Sinagra et al., 1997).It has been extensively used with parasites isolated from sandflies or human MCL lesions to reproduce the clinical manifesta-tions of metastatic leishmaniasis (Travi et al., 1988). Differencesin disseminative propensity were found between various speciesand individual strains (Martinez et al., 1991). In particular, aninfective strain of L. (Viannia) guyanensis (WHI/BR/78/M5313)isolated from a sand fly was shown to be highly metastatic. Clonedlines of this strain, however, also showed a graded metastaticability in the hamster. Thus, allowing for the characterizationof highly (M+), moderately and non-metastatic (M−) para-sites within the same clone. These graded phenotypes remainedstable over several passages (Martinez et al., 2000) and werecaused by varied intensities in inflammatory response (Travi et al.,1996). A decade later, we acquired these same strains, finding theLRVhigh/LRVlow parallel to their M+/M− capability (Ives et al.,2011): the correlation forming the basis of this review. Since then,a different L. guyanensis strain (M4147) derived from a humanlesion has also shown metastatic ability in the hamster model (Reyet al., 1990) and we were able to achieve an identical metastaticrelationship between LRV+/LRV− clones.

Although the development of secondary lesions is rarely seenin murine models of MCL, the LRV-based virulence of these par-asites seems to be reflected in an increased disease severity at theprimary site of infection (Ives et al., 2011; Ronet et al., 2011).Other reports have shown varying results: in BALB/c mice, forexample, M4147 L. guyanensis did not induce progressive lesions(Sousa-Franco et al., 2006) and only very low number of para-sites could be recovered at the site of infection. In these footpads,






there was a well-preserved inflammatory cell population andintact tissue architecture. In the same model, L. panamensis para-sites behave like most Leishmania species inducing a non-healingfootpad swelling (Rojas et al., 1993; Goto et al., 1995) havingthe expected cytokine profile of a “losing battle” in the draininglymph nodes. Here, an early induction of IFN-γ and minimallydetectable IL-4 at 24 h post infection was followed by a completereversal 7 days post infection (Guevara-Mendoza et al., 1997).Whether these strains were infected by LRV is unknown but thusfar, LRV has not been identified in L. panamensis (although asnoted earlier, this species has been less thoroughly screened).Regardless, it is likely that the presence of LRV in L. guyanensisand L. braziliensis strains is only one of the mechanisms con-tributing to disseminated and metastatic leishmaniasis.

Our study on the TLR-dependent recognition of LRV in theL. guyanensis M5313 strain was continued in TLR3−/− andTLR7−/− murine models. As anticipated, the lack of TLR3 sig-nificantly decreased footpad swelling and diminished parasiteload whereas no distinguishable difference in disease phenotypewas observed in mice infected with M- (LRV1low) parasites orbetween WT and TLR7−/− infected mice with either parasite iso-late. We could conclude that these TLR3-dependent responsesto M+ (LRV1high) parasites resulted in elevated disease severityin mice and provide evidence that LRV1 within metastasizingL. guyanensis parasites promotes inflammation, and is impli-cated in susceptibility to infection. Our mouse work correlatesLRV1 presence in parasites with the hyper-inflammatory immuneresponses characteristic of MCL disease. This is the first descrip-tion that TLRs can contribute to Leishmania susceptibility (andnot resistance). It remains to be determined how representativethe mouse model is of the involvement of LRV in disease pathol-ogy in humans. Although the current mouse model does notdisplay all of the hallmarks of MCL, it provides many advantagesover the hamster, specifically in the broader availability of knock-out varieties and reagents for an immune response that is muchbetter described.

L. braziliensis has greatly differing phenotypes in either ani-mal model. In the hamster, it was shown that secondary viscerallesions could arise from a primary CL and also that CLs couldappear subsequently to primary visceral ones (Almeida et al.,1996). While infections in BALB/c mice only gave rise to small,transient footpad lesions (Dekrey et al., 1998; Lima et al., 1999)and further, require a high dose inoculum (1 × 107) to gen-erate this weak response. Similarly in an ear dermis model ofL. braziliensis (De Moura et al., 2005) [based on an existing modelin L. major (Belkaid et al., 1998)], lesions were minor and tem-porary (De Moura et al., 2005), albeit that parasites persisted inthe draining lymph nodes (De Moura et al., 2005; Rocha et al.,2007). The differences in disease outcome may be formed atthe level of the immune response. IFN-γ produced by CD4+and CD8+ T cells was shown to be important (Dekrey et al.,1998; De Souza-Neto et al., 2004; De Moura et al., 2005; Rochaet al., 2007) concomitant with the expression of a broad spectrumof chemokines attracting neutrophils, monocytes/macrophages,NK, CD4+, and CD8+ T cells (De Moura et al., 2005; Teixeiraet al., 2005). It is also interesting to note the Th2 cytokine IL-4 wasonly present at low levels during the first 3 weeks of L. braziliensis

infection, becoming undetectable from day 42 post infection(Dekrey et al., 1998).

The factors underlying the presence or absence of metastaticlesions in animal models are, as yet, unknown. For the human,however, it has been suggested that quiescent or slow-growingparasites could be reactivated and metastasize to mucocutaneoussites following immuno-suppressive treatment (Motta et al.,2003) or during in stress situations (Travi et al., 1988, 1996) whichare perhaps not encountered in the laboratory model.

HOST GENETIC FACTORS IN MCLAt the genetic level in humans, alleles encoding TNF-α, TNF-β,IL-6, CXCR1, and CCL2/MCP1 were associated with an increasedrelative risk of MCL (Cabrera et al., 1995; Nashleanas et al.,1998; Castellucci et al., 2006, 2010; Ramasawmy et al., 2010).Some examples of polymorphisms identified in patients withMCL include (1) a homozygous polymorphism in intron 2 ofTNF-β, (2) a single base pair substitution at position -308 in thepromoter for TNF-α, and (3) a single G-to-C base pair substitu-tion at position -174 in the promoter for IL-6 (Blackwell, 1999;Sakthianandeswaren et al., 2009). Additionally, in VenezuelanMCL patients, there is a preferential expression of HLA class IIDQw3 (Lara et al., 1991).

The presence of LRV and its dsRNA acting on TLR3 could shedlight on other possible genetic polymorphisms in the host. Theroles of TLR3 in the metastasis of carcinomas has already beendescribed, where it was shown to both prevent as well as underliemetastatic processes and their tropism to nasopharyngeal tissue.Here, TLR3 activation inhibited metastasis via down-regulationof the chemokine receptor CXCR4 (Zhang et al., 2009) but hassince been shown to have an opposing role, promoting metas-tasis (Matijevic and Pavelic, 2011). Similarly, it is also possiblethat the effect of LRV can be differentially modulated furtherdownstream of its TLR3-dependent recognition, for example inthe induction of IFN-β and production of CCL5, CXCL10, IL-6,and TNF-α. The balance between pro- and anti-inflammatorycytokines and chemokines can be shifted by polymorphisms inTLR genes (Kutikhin, 2011), increasing the risk of chronic inflam-mation and infection. If a predisposition to the disease can befunneled down to single immune mediators, it may pave the wayfor a new, immunomodulatory approach to the treatment andprevention of metastatic leishmaniasis.

CURRENT THERAPIESCurrent therapeutic strategies in leishmaniasis are far from satis-factory. The growing demand for new anti-leishmanial drugs isparallel to a spreading drug resistance across the genus as wellas their variable efficacy and toxicity in different patients (Croftet al., 2006). A major problem exists in the immunocompro-mised population, often co-infected with HIV and following analready toxic regimen of hepatically metabolized treatments. Themost popularly used drugs in the management of leishmaniasesare based on the pentavalent antimonials, sodium stibogluconate(Pentostam), and meglumine antimoniate (Glucantime) (Harderet al., 2001). Amphotericin B, a polyene antibiotic, has been usedas a second-line treatment for leishmaniasis since the 1960s. It isproposed to work by encouraging a Th1 immune response and






inducing cytokines such as TNF-α and IL-1β as well as generatinga parasite-killing respiratory burst (Wolf and Massof, 1990; Vonket al., 1998; Cuna et al., 2007).

MCL development is, so far, impossible to predict andexceedingly difficult to treat; often displaying drug resistanceand predisposing the host to opportunistic infections. Patientsare frequently refractory to antimony treatment and in extremecases, a combination of antimony and amphotericin B is requiredbut even then, treatment failure is observed. Although antimonyresistance has been proposed to cause these relapses, the factorsunderlying MCL development and its subsequent treatment fail-ure are unknown. Sensitivity to antimony treatment might alsodepend on intrinsic factors within the infecting L. (Viannia) par-asites (Arevalo et al., 2007; Souza et al., 2010). Whatever the casemay be, the limited efficacy and major side effects of antimonytreatment expose a clear need for the development of new drugsspecifically designed to treat MCL. These drugs should take intoaccount the intrinsic variations of the metastatic parasite as well asthe complex inflammatory processes shown to be the root causeof this outcome.

Indeed, controlling inflammation could be an alternative tocomplement conventional drug therapies. Already, interestingresults have been reported for the use of the anti-inflammatorydrug tamoxifen in MCL patients (Miguel et al., 2009). Further,treatment with the anti-inflammatory TNF-α inhibitor, pentoxy-phylline in combination with antimony was shown to be effectivein MCL patients unresponsive to antimonial therapy alone (Lessaet al., 2001). Other immunomodulatory drugs have been sinceproposed such as thalidomide (Blackwell, 1999). However, anti-inflammatory drugs in leishmaniasis should be used with caution,especially when there is no evidence of hyper-inflammation. Thisis because anti-inflammatory or immunosuppressive agents canresult in the reactivation of leishmaniasis as seen in leishma-nial patients treated with anti-TNF-α for rheumatoid arthritis(Franklin et al., 2009).

Miltefosine, an oral drug effective against VL has been testedin MCL. Surprisingly, L. braziliensis parasites were more resis-tant to miltefosine than L. donovani (Sanchez-Canete et al., 2009).This difference could be explained by a reduced transport of thedrug through the miltefosine transport complex (Sanchez-Caneteet al., 2009); a situation illustrating the importance of the intrin-sic factors of metastatic parasites in determining the efficacy ofdrug therapies. Other parasite parameters, which could influencethe efficacy of anti-leishmanial drugs, are the variable levels ofresistance to oxidative stress and strain-specific differences on theinnate immune response. Antimony has been shown to activatethe cell death pathway in several Leishmania species by gener-ating oxidative stress in the form of H2O2 and NO (Mehta andShaha, 2006). It was recently confirmed that cTXPNx (an enzymeknown to detoxify oxidative compounds), plays a crucial role inprotecting L. donovani parasites against H2O2 and also by coun-teracting antimony drug response (Iyer et al., 2008). Furthermore,increased resistance to NO in human isolates of L. (Viannia)can be correlated to larger lesions (Giudice et al., 2007) as wellas underlie the poor responsiveness to antimony therapy (Souzaet al., 2010). Thus, there is a great need to improve upon thedisappointing arsenal of drugs for mucocutaneous leishmaniasis

(MCL), which are currently poorly suited to the widely vari-able metabolic and immunological abilities of metastatic para-sites.

CONCLUDING REMARKSThe intracellular parasites of the Leishmania (Viannia) subgenusharbor a unique risk for infectious metastasis and the develop-ment of complicated and difficult-to-treat secondary lesions. Thisrisk was shown to have roots in both intrinsic parasite factors aswell as in the immune response launched by the host, where ahyper-inflammatory over-reaction destroys local tissue and influ-ences the efficacy of anti-leishmanial drugs. MCL is a commonoutcome of parasite metastasis, forming debilitating secondarylesions in the mucosa of the mouth and nose where inflam-mation accounts for much of the morbidity associated with thedisease (Marsden, 1986; Martinez et al., 1991, 1992; Osorio et al.,1998; Herwaldt, 1999). Although heritable polymorphisms havebeen identified in both the MCL host and parasite, the genetic,and epigenetic factors predisposing an L. (Viannia) infection tometastatic complications have not yet been investigated in greatdetail.

Our recent data has emphasized the role of an intrinsic par-asite factor in the devolution of disease i.e., Leishmania dsRNAvirus that, when present in L guyanensis, acts as a potent innateimmunogen, redirecting the immune response of the host byinducing a hyper-inflammatory reaction and possibly triggeringdissemination (Ives et al., 2011; Ronet et al., 2011). Although it islikely that LRV is not the only factor involved, its presence couldexplain differences in the clinical outcomes observed betweenLeishmania species and/or strains and holds great potential as anew target for treatment strategies. Therapeutic possibilities existin either pursuing LRV itself (antiviral therapy) or in reversing theanti-viral immune response it induces. Indeed, drugs counteringthe type of hyper-inflammation caused by LRV have been success-ful in the treatment of MCL. Tamoxifen (Miguel et al., 2009) anda TNF–α inhibitor, pentoxyphylline (Lessa et al., 2001) for exam-ple, were used in combination with antimony and were shown toaid in the resolution of disease. It would be interesting to deter-mine whether these drugs have an independent or supportingrole to antimony, perhaps only working to create an environmentin which antimony is effective. Refractory and secondary MCLlesions often display antimony resistance and drugs reverting thisprocess are obviously much desired.

The possibility that MCL development is caused by a mis-guided immune response against its viral hyperpathogen providesa novel means of diagnosing the metastatic risk of leishmaniasisas well as creating a better understanding of the treatment neededto cure it. The fact that this risk could be caused by a parasiticfactor (as opposed to a human susceptibility) is not surprising, asit would follow the many observations that deviations in parasitephylogeny mirror their clinical ones.

This is the case between evolutionarily distant members (OldWorld vs. New World) as well as between different isolates ofthe same strain: differences, which are echoed in their immuno-genicity. Comparing the LRV-mediated process of metastasiswith existing models of parasite dissemination could eluci-date the mechanisms underlying recurrence and reactivation,






thus creating a much-needed model system of metastaticleishmaniasis.

The discovery of LRV as an innate immunogen altering thecourse of leishmaniasis should motivate further investigation onthe toll of such viral hyperpathogens on other infections. In thecase of metastatic leishmaniasis, it may provide us with a uniqueopportunity to intervene at a clinical level: for the first time,enabling the diagnosis of metastatic risk and providing a uniquetarget for future therapeutic approaches.

ACKNOWLEDGMENTSThe authors are grateful to members of both groups (NicolasFasel and Stephen M. Beverley): Patrik Castiglioni, Matteo Rossi,Florence Prevel, Chantal Desponts, Remzi Onur Eren, LeyderLozano, Kid Kohl, Ricardo Matin, Lon-Fye Lye, Katherine Owens,Suzanne Hickerson, Erin Acino for support and comments. Thiswork was funded by the grants FNRS N◦ 3100A0-116665/1 andIZ70Z0-131421 (NF) and NIH grant AI 29646 (Stephen M.Beverley).

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Conflict of Interest Statement: Theauthors declare that the researchwas conducted in the absence of anycommercial or financial relationshipsthat could be construed as a potentialconflict of interest.

Received: 25 April 2012; paper pend-ing published: 24 May 2012; accepted:27 June 2012; published online: 12 July2012.Citation: Hartley M-A, Ronet C,Zangger H, Beverley SM and Fasel N(2012) Leishmania RNA virus: whenthe host pays the toll. Front. Cell. Inf.Microbio. 2:99. doi: 10.3389/fcimb.2012.00099Copyright © 2012 Hartley, Ronet,Zangger, Beverley and Fasel. This isan open-access article distributed underthe terms of the Creative CommonsAttribution Non Commercial License,which permits non-commercial use, dis-tribution, and reproduction in otherforums, provided the original authorsand source are credited.


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Leishmania RNA virus: when the host pays the toll